Single photon spectrometer

ABSTRACT

A fiberized single photon sensitive spectrometer on a 32-channel PMT sensor is highly sensitive with broad detection dynamic range. The spectrometer enables accurate and high speed detection, identification and analysis of biological samples labeled with multiple fluorescent markers, such as compositions of multi-color fluorescence signals or radiation emitted by multiple fluorescence dyes. A fiberized optical input of the spectrometer allows an easy and efficient coupling to any measurement system based on fiber collection of the analyzed fluorescence. The spectrometer provides highly accurate DNA sequencing. A 32 channel PMT single photon detector has a detection dynamic range of more than 20 bits and has a frame rate of about 3300 frames per second. The dynamic range of the detector&#39;s pixels reaches 10 8  photocounts per second.

CLAIM OF PRIORITY

This application claims the priority of U.S. Provisional ApplicationSer. No. 61/000,320, filed on Oct. 25, 2007.

The invention disclosed herein was made with Government support underfunding source Award Number R21HG00371702 from the National Human GenomeResearch Institute. Accordingly, the U.S. Government has certain rightsin this disclosure.

FIELD OF THE INVENTION

The present disclosure relates to sensor systems and, in particular, tosingle photon sensor systems and methods for detecting multi-colorfluorescence radiation from, and analysis of, biological samples labeledwith multiple fluorescent markers. The sensor systems and detectionmethods include an optical spectra separation unit, a detection unit andsignal processing algorithms for collected data.

BACKGROUND OF THE INVENTION

A number of fluorescence detection techniques are available based onregistering single photons. Such techniques are commonly referred to assingle photon detection (SPD) techniques. Because of their complexityand cost, in biomedical applications single-photon detection techniquesare mostly used for time resolved fluorescence spectroscopy or detectionof single fluorescent molecules.

As shown in the block diagram of FIG. 1, the conventional single photondetector heretofore consisted of several modules including a singlephoton sensor or photo-multiplying tube (PMT) detector, a pulseamplifier, a pulse shaper, a counter, and a computer. A factor impedingthe performance of single photon detectors heretofore is the relativelynarrow linearity dynamic range of available photon counting devices. Adetector's dynamic range is determined by the response time,τ_(RESPONSE), needed for the detector to respond to a single photon. Thequantity τ_(RESPONSE) depends on the response times of all of themodules of the device and is generally considered to be the sum ofresponse times of the individual modules. As shown in the graph of FIG.2, in practice the shortest response time in currently available singlephoton sensors is about 1 ns. However, conventional single photon PMTdetectors with a given τ_(RESPONSE), have heretofore been limited to adynamic range of 10⁶-10⁷ photocounts per second.

In biological applications, and in particular in DNA detection, DNAsequencing systems have heretofore been unable to process and detectultra-high speed DNA sequencing. In fact, conventional DNA sequencershave been limited to a recording sequencing process at 10-30 frames persecond. Moreover, the dynamic range of conventional DNA machines hasbeen limited to 16 bit. Conventional equipment for serial dilutions ofBigDye DNA sequencing standard have heretofore been relativelyinsensitive by at least a factor of 10 less than what is desirable.

Accordingly, there is a need for sensors including single photondetectors with photon counters having linearity range exceeding 10photocounts per second, for example up to 10⁸ photocounts per second,DNA sequencing processing capability of greater than 10-30 frames persecond and a far more sensitive BigDye DNA sequencing standard.Spectrometers based upon multi-channel single photon detectors, forexample based upon a 32-channel PMT sensor, would enable a veryaccurate, high speed detection of multi-color radiation. In particular,such detectors would demonstrate highly accurate and fast recognition ofcombinations of fluorescent moieties, and high quality detection of DNAsequencing.

SUMMARY OF THE INVENTION

A fiberized single photon sensitive spectrometer based on a 32-channelPMT sensor is highly sensitive with broad detection dynamic range. Thespectrometer enables accurate and high speed detection, identificationand analysis of biological samples labeled with multiple fluorescentmarkers, such as compositions of multi-color fluorescence signals orradiation emitted by multiple fluorescence dyes. A fiberized opticalinput of the spectrometer allows an easy and efficient coupling to anymeasurement system based on fiber collection of the analyzedfluorescence. The spectrometer provides highly accurate DNA sequencing.A 32 channel PMT single photon detector has a detection dynamic range ofmore than 20 bits and has a frame rate of about 3300 frames per second.The dynamic range of the detector's pixels may reach 10⁷ photocounts persecond and can be enhanced by a factor of 10.

Signal processing methods are employed which effectively increase thedynamic range of multi-channel detectors to enable detection andrecognition of combinations of multiple fluorescent moieties. In oneembodiment, a fluorescence detecting sensor is described which is ableto measure single photon radiation emitted by mixtures of minute amountsof multiple fluorescence dyes and very accurately determine the contentof individual dyes in a dye mixture. Such a sensor, or single photondetector, including a 32 channel PMT, a pulse amplifier, comparator, andcounter may have τ_(RESPONSE) times equal to or smaller than 1 ns, forexample 0.1 ns or 0.01 ns. Signal processing algorithms are utilizedwhich enable an accurate separation of fluorescence signals emitted byindividual fluorescence dyes.

In particular, the embodiments herein disclose:

Optical fibers communicating polychromatic light to a light spectraseparator;

At least one multichannel photosensor, each photosensor channel havingphotosensitive pixels adapted to receive distinct light spectra fromsaid light spectra separator and to produce current pulses in responseto single photons of said received light spectra;

A multichannel amplifier, each amplifier channel adapted to receive saidcurrent pulses corresponding to light spectra from a corresponding oneof said sensor channels of said multichannel photosensor and to amplifysaid current pulses; and

A multichannel photon counter, each counter channel adapted to receivesaid amplified current pulses from a corresponding one of said amplifierchannels of said multichannel amplifier, said multichannel photoncounter having an integrator adapted to sum said amplified currentpulses in each counter channel over a predetermined time interval.

There is also disclosed a method for identifying DNA sequencescomprising the steps of:

Labeling selected DNA fragments with fluorescent dyes;

Inputting said DNA fragments to an optical fiber separation capillary;

Illuminating said labeled DNA fragments in said optical fiber separationcapillary with laser light of a predetermined wavelength to producefluorescence spectra from said DNA fragments;

Illuminating optical fiber with the fluorescence spectra from said DNAfragments, said optical fiber conveying said fluorescence spectra to alight spectra separator, the output from said light spectra separatorbeing incident upon at least one multichannel photosensor, eachphotosensor channel having photosensitive pixels adapted receivedistinct light spectra from said light spectra separator and to producecurrent pulses in response to single photons of each differentwavelength of said distinct light spectra.

There is further disclosed a method for detecting color encodedmicroparticles comprising the steps of:

Labeling microparticles with fluorescent dyes;

Suspending said labeled microparticles in a buffer fluid;

Passing said buffer fluid with the labeled microparticles through anoptical fiber capillary at a predetermined speed;

Illuminating said labeled microparticles in said optical fiber capillarywith laser light to produce fluorescence spectra therefrom;

Illuminating optical fiber with the fluorescence spectra from saidlabeled microparticles, said optical fiber conveying said fluorescencespectra to a light spectra separator, the output from said light spectraseparator being incident upon at least one multichannel photosensor,each photosensor channel having photosensitive pixels adapted receivedistinct light spectra from said light spectra separator and to producecurrent pulses in response to single photons of each differentwavelength of said distinct light spectra.

BRIEF DESCRIPTION OF THE DRAWINGS

For a further understanding of the present invention, reference may behad to the following drawings in which:

FIG. 1. is a block diagram of a conventional single photon sensor;

FIG. 2. is a graph showing pulses measured directly from one channel ofa commercial 32-channel PMT;

FIG. 3 is a block diagram of a sensor having single photon sensitivity;

FIG. 4 is a schematic diagram of a spectral separation module;

FIG. 4A is a perspective drawing of the spectral separation module ofFIG. 4;

FIG. 5 is a perspective drawing of a double spectral separation modulehaving two 32-channel PMT detectors;

FIG. 6 shows pulses measured from a SiPM diode after amplification witha 400 MHz 26 dB amplifier;

FIG. 6A is an electronic circuit diagram of a 3-stage, 2 GHz, 60 dBpulse amplifier;

FIG. 7 illustrates a typical 1 ns voltage pulse from a PMT;

FIG. 8 is a schematic block diagram for a 32 channel single photondetector;

FIG. 9 depicts the pulse shape of amplified pulses from one channel of a32 channel PMT and pulse shape after a PECL comparator;

FIG. 10 is a circuit diagram of a 2-stage pulse amplifier;

FIG. 11 is a graph showing channel crosstalk of a single photondetector;

FIG. 12 is a block diagram of a high speed photon counter;

FIG. 13 is a graph showing light vs. photocount characteristics of onechannel of a single photon detector;

FIG. 14 illustrates channel resolution in the top panel and wavelengthresolution in the bottom panel of a spectrometer with a 32 channel PMTsensor;

FIG. 15 is a block diagram of an optical system for a single photondetector;

FIG. 16 is a graph showing light vs. photocount characteristics of onechannel of the single photon detector of FIG. 15 measured for threewavelengths;

FIG. 17 illustrates histograms of photocount distributions at the outputof a photon detection module;

FIG. 18 consists of a photograph and schematic diagram of a single laneDNA sequencer;

FIG. 19 consists of a graph showing the system matrix C in the top paneland color deconvolution matrices in the bottom panel used for processingDNA sequencing data;

FIG. 20 are graphs of DNA sequencing traces and base calling qualityscores;

FIG. 21 consists of graphs showing recognition of color codes andaccuracy of computer simulated color decoding;

FIG. 22 is a schematic drawing of a bead detection system;

FIG. 23 is a graph showing recognition of randomly colored beads;

FIG. 24 is a block diagram of a 32 channel fast, large dynamic rangesingle photon spectrometer;

FIG. 25 is a schematic circuit diagram for a 32 channel pulse amplifier.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

With reference to FIG. 3, there is shown a block diagram of a sensor 10having single photon sensitivity for measuring radiation emitted bymixtures of minute amounts of multiple fluorescence dyes. An opticalinput fiber 1 collects polychromatic fluorescence which is then passedthrough a spectral separation module 12, schematically illustrated inFIG. 4. After passing through the module 12, the decomposed fluorescentsignal illuminates photosensitive pixels of a 32-channel photo-sensor13, which may be, by way of example only, the 32-channel PMT arrayH7260-20, which is manufactured by Hamamatsu, Corporation of Japan. Eachof the separated wavelengths is detected by one channel of the PMT andeach channel of the PMT is capable of detecting wavelengths in the rangeof about 10 nm. The received photons produce very short current pulseswhich undergo amplification and photon counting. For example, as shownin FIG. 7, when the PMT is working in a single photon counting mode eachchannel produces a stream of short, about 1 ns, current pulses inresponse to an incident photon flux. The pulse amplitudes range between0.4-0.6 mA with corresponding peak voltage between 8 and 12 mV.

The pulses are counted by a comparator which is set at a thresholdvoltage smaller than the pulse amplitude. The obtained photocount istransferred to a computer 14 for recording and data processing.

In one embodiment, the spectral separation module 12 performs separationand measurement of polychromatic fluorescence within a range ofwavelengths from 480 nm to 630 nm. With reference to FIGS. 4 and 4A, thepolychromatic fluorescence is presented to the spectral separationmodule through an optical fiber input 16 coupled to a collimator 17, anexample of which may be the F810SMA-543 collimator, manufactured byThorlabs Inc, NJ. The collimator 17 produces a parallel polychromaticbeam of about 10 mm in diameter. The collimated or parallel beam passesthrough a laser rejection tilter 18 and undergoes separation into itsconstituent wavelength components by a diffraction grating 19, which maybe a diffraction grating such as diffraction grating GR13-1850,manufactured by Thorlabs Inc, NJ. In one embodiment, the diffractiongrating 19 may be tuned to perform spectral separation and measurementof fluorescence within the range of wavelengths from 490 nm to 630 nm.

The separated monochromatic beam components are focused onto pixels ofthe 32-channel PMT 13. Such focusing may be accomplished by a sphericalfocusing mirror 21, which may be the spherical mirror CM254-075-G01,manufactured by Thorlabs Inc, NJ and a cylindrical lens 22, which may becylindrical lens LJ1095L2, also manufactured by Thorlabs Inc.

In one embodiment, the spectral separation module is capable ofdetecting wavelengths which differ by 10 nm. Each separate wavelength ismostly detected by one channel of the PMT. Each PMT channel detectswavelengths in the range of about 10 nm.

The spectral separation module of the spectrometer can provide spectralresolution as high as 1 nm. Spectral resolution of about 10 nm may beobtained by a 32-channel PMT having 0.8 mm×7 mm detection zonesseparated by 0.2 mm distance. It has been found that the overallspectral resolution of the sensor can be improved by using arrays ofphotoreceiving fibers, each array being connected to illuminate a singlephoton sensor. The spectral resolution of a spectrometer with a fiberbundle for each photon sensor has been found to be about 5 nm. In fact,the receiving fibers can be used as band pass filters in someapplications when high spectral resolution is required. Wherefluorescent dyes have a wide optical spectrum, several arrays of fiberbundles may be used to collect the dye spectrum and to direct it tocover several channels of the spectrometer, as described in more detailbelow.

With reference to FIG. 5, one embodiment of a spectrometer may beadapted for a wider range of wavelengths. To increase the range ofwavelengths a dichroic plate beam splitter 23 may be used and, as anexample only, may be the beam splitter NT47-424, manufactured by EdmundOptics, Inc, NJ. The beam splitter may also be the beam splitterFF650-Di01, manufactured by Semrock Inc., NY. After the incidentradiation, or light, is collimated the beam splitter 23 divides thecollimated light into two parts. The shorter wavelengths, e.g. 530-585nm for an Edmund beam splitter or 500-640 nm for a Semrock beamsplitter, are deflected into one path 24. At the same time, the longerwavelengths, e.g. 601-800 nm for an Edmund beam splitter and 660-825 nmfor a Semrock beam splitter, are directed into a second path 26. Thelonger and shorter wavelengths are thereby spatially separated byapproximately 90 degrees within the spectrometer. The use of a dichroicplate beam splitter permits a wider range of spectral separation andbetter spectral resolution in the spectrometer within wavelength rangesof 500-800 nm and 300-500 nm, depending upon the type of detectorequipment. Commercially available detector models H7620-20 and H7620-04have been found useful, respectively, for such wavelength ranges.

Persons of ordinary skill in the relevant art will understand that asensor or spectrometer may include two different types of multi-channelsingle photon detectors. For example, without departing from the scopeof the invention, a PMT may be used for the longer wavelengths and asilicon photomultiplier, SiPM, diode detector may be used for theshorter wavelengths. The SiPM diode is CMOS technology, relativelyinexpensive, and consists of a matrix of individual pixels connectedtogether in parallel on a common silicon substrate. In response tosingle photons a SiPM produces single voltage pulses. The technology issuch that arrays of SiPMs may be used with the driving and read-outelectronic circuit integrated on the same chip. SiPMs are high gain, onthe order of 10⁵-10⁷, and operate on relatively low voltage, for exampleon the order of 20-70V. Their response time varies in the range of 1-20ns. With reference to FIG. 6, it may be seen that about 2 ns singlephoton pulses may be obtained from a SiPM diode after amplification witha 400 MHz, 26 dB pulse amplifier. It has been found that 1-stage,2-stage or 3-stage 2 GHz, 60 dB SiPM pulse amplifiers in conjunctionwith an SiPM diode can be used as a detector for single photons insingle photon counting regime. FIG. 6A shows an electronic circuitdiagram for a 3-stage, 2 GHz, 60 dB pulse amplifier. SiPM arrays mayalso be used as a single photon sensor for a single photon spectrometer.SiPMs have also been found to be adaptable to the detection of DNAsequencing.

It will be understood by those persons of ordinary skill in the relevantart that a semitransparent mirror may be used in place of a dichroicbeam splitter. The use of a semitransparent mirror may increase thedetection dynamic range since the entire photon flux received by thespectrometer will be detected by two single photon detectors. Similarly,the photon flux may be split into several fluxes each such flux to bedetected by a dedicated single photon detector. Such an approach alsocan be used to increase a dynamic range of the photon detection system.

With reference to FIG. 8, in one embodiment a detector consists of a 32channel pulse amplifier 27 that may be based on surface mounted devicetechnology and a 32 channel photon counter 31 that may be based on afield programmed gate array. The amplifier portion of the detectorcontains 32 identical pulse amplifying channels 28 each of which, in oneembodiment, has 35-40 dB gains and 1 GHz bandwidth. The detector mayalso be provided with 32 fast comparators 29 each having rise and falltimes of about 2 ns. This arrangement limits the minimum pulse width toapproximately 4.5 ns and increases PMT negative pulses from 10-50 mV tolevels that can reliably trigger the comparators. FIG. 9 depicts in thetop trace the pulse shape of amplified pulses from one channel of a 32channel PMT and in the lower trace the pulse shape after a PECLcomparator.

With reference to FIG. 10, which depicts an embodiment of an electroniccircuit for a pulse amplifier, a 2-stage amplifier consists of 2 BGA 427amplifiers 32 and 33, namely U57 and U58 respectively. Each amplifierstage provides 20 db amplification in the range of 2 GHz. The amplifiedsignal is input to an ADCCMP553 LVPECL comparator 34 having aτ_(RESPONSE) of 1 ns. A negative amplified signal pulse generates aPECL-level output pulse from the comparator, which is input to thecounter 31 (FIG. 8).

The reference voltage can be adjusted using a potentiometer 36, namelypotentiometer R9, from 0 to 3.3 volts. In one embodiment, the referencevoltage can be set from 1.2V (the midpoint of the amplifier) up to theamplified pulse height. A threshold voltage can be selected to be asclose to the pulse bottom as possible while staying above the noiselevel. It will be understood that a I-stage amplifier may also providesufficient gain for triggering the comparator 34. It such an embodiment,the capacitor 37, namely capacitor C35, may not be used. The resultingcircuit provides 20 dB amplification and changes polarity of the pulseonly once. As indicated, FIG. 9 depicts in the top trace the pulse shapeof amplified pulses before being input to the comparator 34 and in thelower trace the pulse shape of the pulses output from the comparator 34.

Two major types of cross talk may occur in a single photon detector. Onetype is electronic cross talk inside the 32 channel PMT. FIG. 11graphically represents typical crosstalk between the neighboringchannels of the PMT. Such cross talk arises by certain features of theelectronic optics inside of the PMT. The channel cross talk in a32-channel PMT contains an optical and an electronic component. Theoptical cross talk arises from illumination of neighboring channels dueto imperfect light focusing, and the electronic cross talk is caused byinternal electron fluxes between the PMT channels. Minor channel crosstalk on the order of 3 percent, may be observed within known 32-channelPMT's when individual channels are illuminated by optical fiber.

The other type of cross talk is electronic cross talk between theamplification channels in the amplifier and the channels in the counter.Generally, electronic cross talk between channels of the amplifier andchannels of the 32 channel photon counter has not been observed.However, even very small channel cross talk in the sensor may cause anambiguity in, for example, the analysis of dye mixtures, particularlywhen the composition of different dye components differs by orders ofmagnitude. In order to minimize the optical crosstalk, single PMTchannels may be illuminated by focusing on them a beam from a commercial532 nm NdYAG laser. As can be seen, the entire channel crosstalk islimited to a few percentage points and it is linear relative to thesignal measured in the main channel.

Referring again to FIG. 8, after amplification the output from each ofthe channels of the amplifier 27 is input to the 32 channel high speedphoton counter 31. The counter 31 sums the pulses arriving at thechannel inputs. The integration time intervals are provided by asynchro-generator in the counter 31 (not shown). In one embodiment, theminimum integration time is about 0.3 ms.

With reference to FIG. 12, a block diagram provides detail of anembodiment of the high speed counter 31 (FIG. 8). In this embodiment thehigh speed counter comprises a pair of identical counting circuits 38and 38A. Each counting circuit consists of a three-byte PECL counter 39and 39A, three PECL to TTL 3-state buffers 41A-C and 41D-F respectively,and three AND gates 42A-C and 42D-F respectively. Each counting circuitis input to a synchronization circuit consisting of a fixed valuesync-byte unit 44, a sync-blocks two-byte counter 46, three 3-statebuffers 47A-C and 3 AND gates 48A-C.

The high speed counter also contains a control circuit 49 which consistsof a crystal oscillator 51, a 14-stage binary ripple counter 52, and aplurality of switches 53 each of which corresponds to a stage of thecounter 52. The control circuit 49 also includes a bytes counter 54, theoutput of which is input to a blocks counter 56. The bytes counter 54and blocks counter 56 are each connected to the AND gates 42A-F and48A-C. The output of the bytes counter 54 is also input to a pair ofadditional AND gates 57A and 57B each of which is connected to one offour TTL to PECL converters 58A-D. A NOT gate 59 is connected to theblocks counter 56 and the AND gates 42A-F and 48A-C. The control circuitcontains a flip-flop connected to each of the four TTL to PECLconverters 58A-D. An LPT output control circuit 62 consists of a timedelay unit 63, OR gates 64 and a flip-flop.

Pulses from the high speed amplifier 27 are simultaneously provided tothe inputs of the 3-byte PECL counters 39 and 39A. One of these countersis always in counting mode, while the other is on hold. Accumulated datais transferred byte after byte to an LPT port 67 through the PECL to TTL3-state buffer that corresponds to the byte number.

The states of the three-byte PECL counters 39 and 39A are determined bythe control circuit signals converted by corresponding TL to PECLconverters 58A-D. As an example, the output signal from the TTL to PECLconverter 58B sets and holds the three byte PECL counter 39 in the holdmode and, at the same time, sets the three-byte PECL counter 39A intothe counting mode. The output signal from the TTL to PECL converter 58Csets and holds the three byte PECL counter 39A into the hold mode andsets the three-byte PECL counter 39 into the counting mode. The outputsignals from converters 58A and 58C reset the corresponding three-bytePECL counter after its data has been transferred to the LPT port 67.

After each 102 transmitted bytes three synchro-bytes from thesynchronization circuit 43 are initiated. The 1^(st) byte has the fixedvalue of 00010001, as indicated on FIG. 11, and the 2^(nd) and 3^(rd)bytes represent the then current state of the sync-blocks two-bytecounter 46. The synchro-byte output of the counter 46 is passed to thesystem output 67 through the corresponding 3-state buffers 47A-C.

The crystal oscillator 51 of control circuit 49 generates 2 MHz clockpulses which are passed to the 14-stage binary ripple counter 52. Thecounter 52 has 14 output pins, indicated generally by reference numeral68. Each pin outputs clock pulses the initial frequency of which isdivided by a coefficient from 2 to 16386. The switches 53 are used toselect an appropriate pin from 1-14 and thereby set the clock frequencyfor the entire circuit. In this embodiment, the switches can set thetransmission speed of the data to the LPT port 67 from 122 to 1,000,000bytes per second.

The clock pulses are passed to the bytes counter 54. After receivingeach clock pulse the bytes counter 54 sequentially generates a signal oneach of three outputs, indicated as “Byte” numbers 1, 2 and 3 on FIG.11. Each such signal transmits a corresponding data byte and each databyte is passed to both counting circuits 38 and 38A. Each data signal isalso presented to the synchronization circuit 43 via a corresponding ANDgate 48A-C.

A high on the 4^(th) output from bytes counter 54 resets the counter andis passed to the blocks counter 56 which counts the number of thetransmitted 3-byte words. Upon counting the 34^(th) 3-byte word, whichequals 102 data bytes, the blocks counter 56 generates the signal forthe 35^(th) 3-byte word. This signal is presented to the AND gates 48A-Cwhich enable the synchronization circuit's data to be presented to theLPT output 67. At the same time, the signal for the 35^(th) 3-byte wordblocks the output of the counting circuits 38 and 38A. The output signalfrom blocks counter 56 that corresponds to the 36^(th) 3-byte word,resets the counter, as indicated on FIG. 11, and clocks the sync-blockstwo byte counter 46 to count the number of 105 byte sequences. Thesync-blocks two byte counter 46 resets after a value of 65536 isreached.

The flip-flop 61 is triggered by every 1^(th) byte pulse from the bytescounter 54. The signals produced by the flip-flop 61 alternately putthree-byte PECL counters 39 and 39A into count or hold modes. Suchsignals also enable or disable the output of the counters and eitherallows or blocks the transmission of the reset pulse to the counters viaAND gates 57A and 57B. FIG. 13 depicts a graph showing the typical lightvs. photocount characteristic of one channel of the type of counterdepicted in FIG. 12.

Referring again to FIG. 7, typical voltage pulses are depicted at theinput and the output of an embodiment of a pulse amplifier. The width ofthe amplified pulse is approximately 5 ns which is about 5 fold longerthan the width of the input pulse. The dynamic range of the detector(the ratio of the maximum photocount to the dark noise) is −20 bit. Withthe use of data recording software, pulse duration less than 2 ns may beresolved by the counter with pulse amplitude −0.5V.

Due to the sensitivity and linearity of the 32-channel single photondetector of the present embodiment, a photocount rate as high as 5×10⁷counts per second can be registered. In fact, the linearity of thedetector enables an extremely broad range of linear photon counting, forexample, up to and exceeding 2×10⁷ photocounts per second. Photon countsas high as 2×10⁸ have also been observed. These count ranges wouldexceed the detection dynamic range of any known commercial single photondetector. Comparison of the photon detection efficiency of the detectorof the present embodiment and an available commercial single photondetector, for example the model SPCM-AQR-12-FC, indicates that at 490 nmthe photon detection efficiency of a 32-channel PMT detector of thepresent embodiment constituted about 20% of the photon detectionefficiency of the foregoing commercial SPCM and it decreased to 5% at610 nm.

With reference to FIGS. 8 and 12, data collected by the counter 31 istransferred to a PC using a standard IEEE 1284 Parallel Port Interface67. The data is transferred in 105-byte frames using a binary format.Data frames consist of count values obtained for each of the 32detection channels (3 bytes per channel). Each frame starts with a6-byte header which includes the following fields: I-byte counter type,2-byte frame number, and a 2-byte counting period length measured inmilliseconds. The frame number contains the number of the current frame.The number is incremented by 1 for each following frame thus forming arising sequence with overflow. The frame numbers serve assynchronization marks and are used by the data processing software tofind data frames in the continuous data stream. Frame numbers are alsoused for verification of data integrity and for finding errorsintroduced by interference in the transmission line. A special softwarepackage performs the recording and the on-line visualization of the datatransferred by the counter.

In processing fluorescence spectra, the main task of the signalprocessing is the determination of contributions of individualfluorescent dyes into fluorescent signal generated by dye mixturescomposed of n dyes having distinct and known fluorescent spectra. Thecontributions of individual dyes in the mixture can be found by adecomposition of the fluorescence measured in N independent channels ofthe spectrometer, provided the spectrometer produces linear response tothe detected fluorescence.

By way of example, if the number of the analyzed fluorescent dyes n<N(e.g. <<=4 for DNA sequencing) then, using a known system matrixtechnique H_((N×n))=(h₁, h₂ . . . , h_(n)), where h_(i)=(h_(1i) . . .h_(Ni))^(T), (1≦i≦n) are N-component vectors representing spectra of thefluorescent dyes in the analyzed dye mixture, it is possible to obtain asystem matrix H by calibrating the system in advance of the spectralresponses for individual fluorescent dyes hi. If r=(r₁ . . . r_(N))^(r)is the measured fluorescent spectrum of the dye mixture, and s=(s₁, s₂,. . . , s_(n))^(T) is a vector of component weights representingconcentrations of individual fluorescent dyes, then in the presence ofnoise ω=(ω₁ . . . ω_(N))^(T) the measured spectrum r is:

r=Hs+ω  (1)

The optimal solution a of Equation (1) depends on the distributionproperties of the noise components ω_(i). A simplified assumption may bemade that ω_(i) is independent and, assuming identically distributednormal random values, a well known and computationally efficient minimumvariance unbiased solution was achieved by Kay in 1993, (Fundamentals ofStatistical Signal Processing. Estimation Theory at p. 97), forestimating ŝ as follows:

ŝ=(H ^(T) H)⁻¹ H ^(T) r  (2)

In photon counting, individual rate observations r_(i) have Poissondistribution with equal mean and variance. For higher photocount rates(over 50 counts per observation period), the observed rates are wellapproximated by superposition of ‘true’ mean rate r _(i) and Gaussiannoise ω_(i) with variance depending on the mean rate as follows:

r _(i) = r _(i)+ω_(i), ω_(i) ˜N(0,f( r _(i)))

More precise solutions can be obtained by g that the components ω_(i)independent non-identically distributed normal random variables. Thegeneral solution for Equation (1) has been derived heretofore by Kay in1993, Id. as follows:

ŝ*=(H ^(T) C ⁻¹ H)⁻¹ H ^(T) C ⁻¹ r  (3)

where C is the co variance matrix of components ω_(i). Due toindependence of ω_(i) the matrix C is diagonal:

$\begin{matrix}{C = \begin{pmatrix}\sigma_{1}^{2} & \; & \; & 0 \\\; & \sigma_{2}^{2} & \; & \; \\\; & \; & \ddots & \; \\0 & \; & \; & \sigma_{N}^{2}\end{pmatrix}} & (4)\end{matrix}$

where σ_(i) ² is the variance of ω_(i). In practice, the mean rate r_(i) is unknown and the observed rate r_(i), is used for the computationof the variance:

r _(i) ≅r _(i).

Variances σ_(i) ² are estimated for each measurement. The function thatrelates σ_(i) ² and r_(i) is specific for each preprocessing method usedto obtain r_(i). For example, if r_(i) are obtained directly by countingof photons during a sampling period, then σ_(i) ²=r_(i). If backgroundb_(i) is subtracted from the result of the counting observation, thenσ_(i) ²=r_(i)+b_(i). If r_(i) is obtained by averaging countingobservation over k sampling periods, then σ_(i) ²=r_(i)/k. The estimatorof Equation (3) is more accurate, but requires more computationalresources than the estimator of Equation (2).

The signal processing technique described above allows backgroundsubtraction at the stage of cross-talk removal. This is achieved bycreating additional spectrum (column in matrix H) that represents thebackground. The estimators of Equations (2) and (3) with new matrix Hwill separate the background from the other spectral components.

With reference to FIG. 15, there is shown a block-diagram of ameasurement setup used for characterization of the single photondetector. In the setup, light from a light source 69, which may be amonochromator or a 532 nm laser, is passed through a set of neutraloptical filters 71 and coupled into the fiber terminated with a GRINlens 72. The light from the GRIN lens is focused onto one of the 32photocathodes of the 32-channel PMT. Photocount and lightcharacteristics measured for the single photon detector using the setupshown in FIG. 15 with a monochromator as the light source 69 aredepicted in FIG. 16. As can be seen, for all three measured wavelengths,the output of the detector remains linear up to 108 photocounts persecond and the detector's dynamic range exceeds 2×108 photocounts persecond. A significant difference in absolute count rate is illustratedfor wavelengths of 488 nm &530 nm as compared to 630 nm. There is adecrease of the quantum efficiency of the PMT at wavelengths longer than600 nm.

The noise in the photon counting system of the present embodiment isdetermined by the temporal distribution of photocounts. A correctlyoperating photon counter utilizes Poisson distribution for which thevariance of the photocount rate estimate, e.g., the number of photonscounted over the integration period, is equal to its mean value. Thissets the lower boundary for the signal-to-noise ratio of the photondetector. FIG. 17 shows the histogram of the photo-count distribution atthe output of the photon detection module for four differentillumination levels. The photocount was collected during 25 ms intervalsand recorded using data recording software for about 30 min for eachillumination level. FIG. 17 illustrates a good match between mean valuesand variances for all four illumination levels. This indicates that themeasured noise is only caused by the stochastic nature of the photonfluxes detected by the detector and that the detector itself does notproduce any additional noise.

In one embodiment, a single photon spectrometer can be used forapplications which require a sensitive detection and recognition ofmixtures composed of several known fluorescent dyes. In particular, thespectrometer described herein may be used as a photo-sensor fordetection of DNA sequencing which is performed by electrophoresis in asingle fused silica capillary using fluorescence excitation withcommercially available Ar-ion and Nd-YAG lasers. In order to suppresslaser wavelengths 50D 522 nm and 538 nm laser edge filters (522AELP,538AELP, Omega Optical Inc, VT, USA) for Ar-ion and Nd-YAG lasersrespectively may be used.

With reference to FIG. 18, there is shown a photograph and schematic ofan embodiment of a single lane DNA sequencer. DNA samples undergoelectrophoretic separation in a single-capillary separation module shownin the photograph. The module consists of a miniature high voltagesupply 73 (up to 15 kV) having a built-in voltmeter and ampere meter, apolymer replacement system 74, a temperature control system 76 (25-70°C.±0.01° C.), a tube-changer carousel 77 for DNA samples and runningbuffer, and a precision optical system 78. With reference to theschematic of FIG. 18, when the fluorescently labeled DNA fragments passthe optical system 78 they are illuminated by a fiberized laser such as,but not limited to, an Ar-ion laser (488 and 514 nm, 20 mW) made byUniphase, CA, or an Nd:YAG laser (532 nm, 25 mW, GCL-025-S) made byCrystaLaser, NV. The laser-induced fluorescence is collected by a fiber81, which may have a 200 mkm core diameter, and is delivered to thespectral separation module 12 (FIG. 3). In one embodiment, 5OD 522 nmand 538 nm laser edge filters (522AELP, and 538AELP) made by OmegaOptical Inc, VT, may be used to suppress laser wavelengths of Ar-ion andNd-YAG lasers, respectively.

DNA samples labeled with the BigDye Terminator v1.1 sequencing standardobtained from Applied Biosystems (Foster City, Calif.) were denatured in25 μl Formamide and diluted 1 to 5 in HPLC grade water immediately priorto injection. DNA separation was conducted at 150 V/cm in 40 cm (35 cmseparation length) uncoated capillaries, having a 50 μm inner diameterand a 365 μm outer diameter, filled with POP-7 separation medium at 50°C. In this example, a running buffer was used.

There are at least two effective approaches for estimating fluorescenceintensities produced by four dyes used for labeling of A, C, G and Tterminators. One such approach is to decompose the measured spectrumaccording to Equation (3), above. With respect to the decomposition ofthe entire measured spectrum, the spectral components c_(i) whichdescribe individual dyes are obtained experimentally by measuringfluorescence emitted by each dye separately and by normalizing themeasured vector of photocount rates. The determination of concentrationsof individual dyes s[n] in the mixture is performed for each time frame.The obtained sequence of dye concentrations forms four sequencingtraces, which are further used for base calling.

Another approach is the application of virtual filters. The method ofvirtual filters may be used if the component spectra are well separated.Incident light containing spectra with spatially well-separatedwavelengths irradiates input ends of fiber bundles having a selectedangle of acceptance. Because of the finite angle of acceptance,particular fibers collect signal only from a specific wavelength anddeliver signal to a specific channel of the photodetector. As anexample, virtual filters may be formed by selecting three PMT channelsso that each of three different wavelengths of the fluorescence of eachdye is captured by one of the three PMT channels and thereforecontributes the most to measurements in the assigned channel andprovides a minimum response in the other channels. Such a system rendersbandpass filters for detection of multicolor fluorescence unnecessary.In another example, spatially wide spectra of a particular dye andhaving a particular wavelength are simultaneously captured by a group ofseveral, for example three, fibers or fiber bundles. Such an arrangementallows a significant increase in dynamic range without a decrease in thesignal-to-noise ratio of the system.

In one embodiment, a high dynamic range of linearity for spectra withnarrow emission spectra may be achieved. There, each of severalwavelengths of the fluorescence of each dye is captured by one of thePMT channels and therefore contributes the most to measurements in theassigned channel and provides a minimum response in the other channels.The fibers pass the specific wavelengths to corresponding detectors in a1×N photodetector. From there, the signals are passed to an M×Nphotodetector for collecting spatially separated spectra. Projectionoptics may be used to project the N_(h) wavelength on the N_(th) columnof the M×N photodetector. Therefore, signals of particular wavelengthsare simultaneously collected by M single photon photodetectors. Thisarrangement also achieves a high dynamic range of linearity and a highsignal-to-noise ratio for the system.

In the data processing approaches for the described embodiments, thefour sequencing traces undergo standard processing which includes noisefiltering or smoothing, baseline subtraction, crosstalk removal,mobility shift correction, peak height and spacing equalization. Afterre-sampling to 7-15 points per peak the traces are stored in .SCF formatand processed by standard base calling PHRED software. The result of theprocessing is returned as a sequence of base-calls with their positionsand quality scores.

As indicated above, there are at least two approaches to processing ofthe DNA sequencing data. With reference to FIG. 19, which illustrates asystem matrix graph 82 and color deconvolution matrices 83 and 84 forAr-ion and Nd-YAG lasers respectively, a simplified data processingapproach would use signals obtained from channel 18 (546 nm), channel 15(568 nm), channel 11 (589 nm) and channel 5 (610 nm) of the PMT.Channels 18, 15, 11 and 5 correspond respectively to G, A, T and C. Thederived color deconvolution matrices 83 and 84 for Ar-ion and Nd-YAGlasers are illustrated in spread sheet format in the lower panel of FIG.19. In contrast, spectral components h_(i) for determination of thesystem's matrix H may be obtained experimentally by measuringfluorescence emitted by each dye separately and by normalizing themeasured vector of photocount rates shown in the upper panel graph 82 ofFIG. 19.

The foregoing approaches to processing of the sequencing data yieldsimilar sequencing traces and base calling quality. However, formeasurements characterized by small signal-to-noise ratios thedecomposition of entire measurement spectrum gives better results sinceit uses the entire information about the fluorescence emitted by eachdye rather than just the fluorescence obtained in only four selectedchannels of the spectrometer. Another advantage of using the entirefluorescence spectrum obtained from the dye is a significant increase ofthe detection dynamic range. Indeed, in the 32-channel photosensordescribed herein the channels have linear response up to 2×10⁷photocounts per second and if the measured fluorescent dye has a broadspectrum, the linear range of the detection for this dye will beproportional to the number of simultaneously illuminated channels of thesensor.

FIG. 20 illustrates two DNA sequencing traces and base calling qualityscores obtained with an embodiment of the spectrometer described hereinusing the Ar-ion and Nd-YAG lasers. The sequencing traces recorded withthe two lasers look very similar and the quality scores are nearlyidentical. The obtained Q20 sequence read length is as long as about 650base pairs which is in a very good agreement with the read lengthsobtained for the same capillary length in commercial DNA sequencers,such as model ABI-3730 from Applied Biosystems Inc.

In one embodiment, the single photon spectrometer may recognize andaccurately decode color codes. With reference to FIG. 21, the upperpanel illustrates measurements and decoding of mixtures containing threetypes of QDs with strongly overlapping spectra. The middle panel of FIG.21 illustrates that when the content mixture of QD2 is varied from 0% to15% a nearly perfect match of the premixed and decoded concentrationscan be achieved. Curves on the bottom panel of FIG. 21 illustrate thedecoding statistics obtained by performing several thousand measurementsfor each QDs mixture (about 10,000 photons per measurement) as well asthe decoding results obtained for computer simulation of themeasurements performed by the single photon spectrometer.

In one embodiment the single photon spectrometer may detectcolor-encoded microparticles. With reference to FIG. 22, there isillustrated a bead reader system in which individual beads are suspendedin a buffer fluid held in a beads' pumping system 86. The beads aremeasured by pushing them through a capillary 87 at a high speed. Anexcitation beam 88 excites fluorescence in the beads. The fluorescenceis detected by an optical fiberized detector 89 and is presented througha suitable spectrometer or optical reflection system 91 to a singlephoton detector 92. The detected signals are recorded by a computer (notshown) and the recorded data is suitably processed. In one embodiment,the pumping system 86 is a programmable micro-pump and the capillary 87is a 25 μm ID capillary. The capillary is inserted into an optical head(not shown) to ensure a uniform illumination of the capillary by theexcitation beam 88. The beam 88 is produced by a suitable fiberizedlaser source (not shown). When the beads pass through the laser beam,they emit fluorescence which is collected by the fiberizedmicro-objective 89. The collected fluorescence is delivered to thespectrometer 91, where it is detected by the single photon detector 92which, in this embodiment, is a high speed multi-channel detector.

With reference to FIG. 23, the bead reader of FIG. 22 may be used tocarry out measurements of randomly colored beads. In one embodiment,measurements were made of polystyrene beads colored with quantum dots.The results of decoding obtained for 100 such beads are illustrated inFIG. 23, where the x axis shows the bead number in the order detected bythe bead reader and the y axis shows the bead codes normalized to 1.

The main characteristic of the foregoing beads' detection system is thenumber of beads that can be detected per unit time and decoded with therequired accuracy. In general, decoding accuracy is determined by thenumber of photons collected during the bead's detection time. It will beunderstood that for commercially available sets of quantum dots,decoding accuracy as high as 99 percent can be obtained if the totalnumber of photons collected from the bead will be larger than 10⁴. Therate at which detection of the beads occurs depends on the dynamic rangeof the photon detector. With reference to the block diagram of FIG. 24,in one embodiment a beads' detection rate as high as 10⁴ per second isobtained with a photon detector consisting of a spectral separationmodule 93, a 32 channel PMT 94, a 32 channel FPGA 96, a high speed USBmicrocontroller 97 and a computer 98. Such a system has a linearityrange of 10⁸ photocounts per second per channel, and a data transferrate of 32 MB per second. A schematic circuit diagram of one channel ofa 32 channel pulse amplifier of the system shown in block diagram inFIG. 24 is illustrated in FIG. 25.

Although various embodiments have been described above with a certaindegree of particularity or precision, or with reference to one or moreindividual embodiments, those skilled in the art could make numerousalterations to the disclosed embodiments without departing from thespirit or scope of the invention as claimed herein. It is intended thatall of the subject matter contained in the above description and shownin the accompanying drawings shall be interpreted as illustrative onlyof particular embodiments and not limiting. Changes in detail orstructure may be made without departing from the elements of theinvention as defined in the following claims.

1.-19. (canceled)
 20. A single photon sensor comprising optical fibersadapted to receive a single wavelength of incident polychromatic lightand to communicate said single wavelength of light to photosensitivepixels defining one or more channels of a multichannel photosensor. 21.The single photon sensor of claim 20 in which said incidentpolychromatic light consists of fluorescent spectra produced by amixture formed by a plurality of fluorescent dyes.
 22. The single photonsensor of claim 21 in which said optical fibers comprise input endsdefining a predetermined angle of acceptance of light corresponding to asingle wavelength of said incident fluorescent spectra.
 23. The singlephoton sensor of claim 22 in which each of said optical fibers isadapted to receive a single wavelength of said incident fluorescentspectra different from the wavelength of incident fluorescent spectrareceived by others of said optical fibers, and to communicate saidsingle wavelength of incident fluorescent spectra to a corresponding oneof the channels of said multichannel photosensor.
 24. The single photonsensor of claim 22 in which a predetermined plurality of said opticalfibers is adapted to receive a single wavelength of said incidentfluorescent spectra, and to communicate said single wavelength ofincident fluorescent spectra to a corresponding plurality of thechannels of said multichannel photosensor. 25.-32. (canceled)